1. Technical Field
The present invention is related to a eukaryotic vector for stable maintenance or expression of DNA sequences or genes in eukaryotic cells. More particularly, the present invention is related to a vector based on the parvovirus, adeno-associated virus (AAV), wherein said vector is packaged into AAV particles. Such particles, when used to infect eukaryotic cells, allow maintenance or expression of DNA sequences or genes at a high frequency. The vector of the present invention is also rescuable from eukaryotic cells
2. Prior Art:
Heretofore, the use of a parvovirus as an expression vector was not known. Other virus-based eukaryotic expression vectors have been described (Eukaryotic Viral Vectors, ed Y. Gluzman, (Cold Spring Harbor Laboratory, N.Y. 1982). However, the other vectors described so far have one or more disadvantages: (a) They do not integrate a foreign gene into the host cell genome at high frequency; (b) are not easily rescuable from the integrated state, or (c) are limited in their host range either for expression or for rescue, or (d) include many other viral genes as well which may complicate analysis of the particular gene under study.
Some studies have shown that a gene inserted into a viral-based vector can be introduced into mammalian cells at high frequency using DNA transfection procedures rather than packaging into a virus particle. However, these transfection procedures result in integration of the gene together with flanking sequences derived from plasmid DNA and generally suffer from the problem of inability to be easily rescued. An additional difficulty with this procedure is that some cells transfect very poorly and indeed some transfection procedures are toxic to certain cell types. In contrast, AAV particles have a high probability of infecting most cell types very efficiently.
Samulski et al., (Proc. Natl. Acad. Sci. USA 1982, 79: 2077) reported construction of a plasmid vector using GC tailing. As would be evident from the detailed description infra of the present invention, the Samulski et al procedure is quite different. Samulski et al., employ GC tailing while the present invention utilizes molecular linkers.
An advantage of the present invention is that AAV can be excised intact, free of plasmid sequence by cleavage in vitro. This is not possible by prior art methods including the Samulski, et al procedure.
Laughlin et al., (Gene, 1983, 23: 65-73) described the construction and cloning of infectious adeno-associated virus (AAV) genomes in bacterial plasmids and speculated on the potential of AAV as a eukaryotic vector. However, an actual rescuable system demonstrating the functional feasibility of an AAV as a eukaryotic vector for introduction and stable expression of genes in eukaryotic cells has not heretofore been known or disclosed.
The present invention for the first time establishes AAV as a system useful as a vector either for transient expression or for site-specific mutagenesis as well as for stable maintenance or expression of foreign DNA sequences or genes in eukaryotic cells. By "foreign DNA sequences or genes", it is meant any DNA sequence other than those in normal wild type AAV.